Cancerous diseases are generally manifested by uncontrolled growth of cells that differ from the normal cells of the afflicted animal. Diagnosis and treatment of cancer have long been problematic. The disease is often far advanced before symptoms are evident, making treatment more difficult. In addition, the biopsy method of ascertaining whether cells are malignant requires surgery to remove a tissue sample and highly trained Pathologists to observe the tissue under a microscope. Treatment of cancer has been hampered by both lack of adequate early detection methods and lack of selectivity of methods employed. Methods effective to eliminate cancer cells may also be harmful to normal cells. Since cancer cells of one type of disease such as leukemia may differ from cancer cells of othter types of disease such as milignant melanoma, treatment could be better tailored to the disease if the technological capability to differentiate cancer cells from normal cells, or among types of cancer cells, existed.
The art has revealed that immunologic differences may exist between cells. The present invention concerns the discovery of an immunologic difference between normal cells and cancer cells that has never before been described and the making of a composition useful for manufacturing antibodies for detecting this difference.
A mammalian immune system generally operates to "respond" to matter that is not part of the normal cells of the organism. This matter is sometimes described as "non-self". One response made by the immune system may be to cause lymphocytes to produce antibodies that can specifically react with sites on the "non-self" matter (antigenic sites). Each unique lymphocyte produces unique antibodies. Each individual antibody molecule may be able to react only with a particular antigenic site. Normal cells may have some antigens that are also found on cancer cells. Therefore one antibody produced by a mouse lymphocyte, for example in response to injection of the mouse with a human cancer cell, could possibly react with both normal and cancerous human cells. Another mouse lymphocyte could simultaneously produce an antibody directed against an antigen on the injected tumor cell that is unique to that tumor cell. This antibody would not react against antigens on normal human cells. Collectively, the mouse's anitbodies in response to the many antigens of the injected cell. It is desirable to isolate only the lymphocyte(s) which produce anti-tumor antibody.
Once appropriate lumphocyte(s) have been isolated, procedures are known for making them into monoclonal antibody "factories". A "monoclonal" antibody is derived from man-made technology in which a single antibody-forming cell is fused with a myeloma cell to form a "hybridoma". A hybridoma has a theoretically infinite life span and produces only one antibody type, each antibody molecule produced being identically specific for a certain antigenic site. With appropriate selection procedures, an anti-tumor antibody-forming cell line may be manufactured. The art has revealed the production of anti-tumor monoclonal antibodies and anti-melanomaassociated antigens via hybridoma techniques.
The present invention concerns the manufacture of a unique hybridoma. The hybridoma of the present invention is a continuous cell line capable of producing a monoclonal antibody, also a subject of the present invention. The monoclonal antibody, which has been named "WI-MN-1", has not been previously described. It has been found to specifically recognize certain antigenic sites of tumor cells, primarily located on melanoma cells. The antigens, never before described, have been isolated and characterized and are also a subject of the present invention.
Prior to the present invention, there has been no report of monoclonal antibodies recognizing these antigens. The monoclonal antibody of the present invention recognizes antigens on cells of some types of tumor tissue but not others. This property makes the monoclonal antibody of the present invention useful for diagnosis of and differentiation of cancerous disease. The specificity of the antibody for tumor antigens and lack of reactivity with normal cells indicates utility for treatment of disease by either antibody injection to counteract tumor antigen or by coupling the antibody to tumor-destroying drug, tumor toxins, or radioactive substances, and allowing the antibody to direct the coupled material to the tumor. Binding a radioactive substance to the antibody is effective in allowing physicians to locate the cancerous cells in the body by using imaging techniques, such as, for example, the gamma camera. The antibody directs the radioactive substance only to certain tumor cell antigens where the radioactive substance emits radiation detectable by non-surgical isotope detection methods. The antibody's specificity also allows for utility in isolation of antigen.
The antigens of the present invention have not been previously described. These antigens can be used in diagnostic tests to monitor natural antibody levels. By elucidating which tumor cells have this antigen, a key to the mechanisms of malignant disease may be found. Because antigens may not be located on the surface of a cell, where they would be amenable to reaction with antibody, during the entire stage of cell life, monitoring the characterized antigen of the present invention may indicate the stage of or progession of disease. The antigen of the present invention has been found to be present on mainly large-sized cells and thus can be a marker for cell size differentiation. Tumor cells are also known to slogh antigens into body fluids, such as the blood, urine, cerebral spinal fluid, peritoneal fluid, pleural fluids or others. The identification and quantitation of an antigen known to be associated with melanoma cells is significant in that it is now possible to monitor the antigen by testing fluids from the body to indicate the size of tumors, as larger tumors would theoretically slough more antigens into body fluids. Also, if a tumor were surgically removed, subsequent monitoring of the body fluids for the presence of melanoma-associated antigens could indicate whether all the tumor was successfully removed. Whether a tumor was successfully eradicated by chemotherapy or radiation could also be monitored by assessing antigen levels. Recurrences of the tumor could be detected by running tests on fluids for the antigen. In such monitoring tests, immunologic (such as enzyme-linked immunosorbent and radioimmunoassay) as well as oteher protein identification methods would be appropriate to identify the melanoma-associated antigen. Demonstration of melanoma antigen, on tissues, can be used to distinguish melanoma from normal tissue or other tumors. The antigen can also be used to elicit a specific antibody response. Rather than injecting an animal with whole tumor cells, which would elicit production of numerous antibodies to tumor and non-tumor antigens, the specific antigen of the present invention can be used to elicit a specific immunological response to known melanoma-associated antigen. Antigens are useful in vaccines to induce the development of immunity. Anti-tumor antibody can be isolated and purified with techniques employing tumor antigen.
The continuous cell line manufactured, which is a subject of the present invention, produces monoclonal antibodies to specific tumor antigen of the present invention. The cell line which has been deposited produces one monoclonal antibody able to recognize the described antigen. Other cell lines may now be easily manufactured because of the present invention which will produce antibodies with the same specificity, and these cell lines are encompassed by the present invention.